JP5185335B2 - Biochips for archiving biological samples and clinical analysis - Google Patents

Abstract

A biochip for diagnostic purposes comprises a sample carrier made of a solid matrix, on the surface of said sample carrier is bound the sample material to be analysed which originates from a biological organism.

Description

  The present invention is suitable for diagnostic purposes and is coated with biological sample material to be analyzed, saving space and archiving sample material and performing laboratory diagnostic analysis on sample material Each is related to a suitable biochip. The invention further relates to a sample carrier suitable for the production of such a biochip and a process for the production of the sample carrier or biochip described above. The present invention further relates to a diagnostic method using the biochip of the present invention and the use of the biochip and diagnostic method of the present invention in various medical fields.

  Laboratory diagnostics are an important basis in therapy. As the number of diagnostic marker molecules available increases, the potential for clinical laboratory diagnosis is constantly expanding. Due to the volume of detection reactions to be performed, the degree of urgency, and economic reasons, automatic analysis methods that can handle many different analyzes in a short time are preferentially used.

  Biomaterials for clinical laboratory analysis of patient health are typically bodily fluids such as whole blood, plasma, serum, urine, ascites, amniotic fluid, saliva, fluid, etc., or tissue samples of different organs. is there. The treating physician potentially collects the sample from the patient by subjecting the patient to a specific treatment, such as centrifugation, and transfers the patient's sample into a test tube that is placed in the test tube It will be sent out or stored until analysis is performed.

  Depending on the parameter to be analyzed, i.e. the component to be analyzed (analyte), the sample is stored at room temperature or stored chilled or frozen. For long-term storage, i.e. when stored for weeks, months or years, patient samples must be frozen at temperatures below -20 ° C to prevent analyte degradation. . The main cause of degradation that occurs at room temperature is the specific enzyme activation in the liquid sample material.

  A typical sample volume for long-term storage is at least 500 μl. If further analytical measurements are to be made at a later time or after a different time after the first analysis, several 500 μl samples are prepared and stored from the original collection of blood, or Must be stored frozen. Because these samples occupy a relatively large amount of space, long-term storage is relatively expensive. For this reason, it is not generally used to store patient samples for long periods of time. However, this loses the possibility of reviewing previously collected samples later. In many cases, especially when it is important to compare a patient's current state to the same patient's initial state, particularly with respect to certain diagnostic parameters, it may be desirable or necessary. In some cases, it may be convenient to record several previous conditions of the patient at various times to perform a trend analysis on the relevant parameters. However, such a comparison is not possible if the relevant diagnostic test has not been or could not be done before and the original sample has not been preserved.

  The most different types of methods are currently used in clinical tests for analysis of patient health. Among these methods, first, there are enzyme activation measurement, special color reaction, immunochemical method, cytological method, and molecular biological method. In recent years, immunochemical methods have become particularly important and have replaced many conventional methods. Molecular biological methods are also emerging in routine diagnostics.

  Currently utilized immunochemical analysis systems are based on antigen-antibody reactions, which are often performed in volumes of about 10-500 μl. Here, a sample of a patient (such as a body fluid) containing the analyte to be detected, in this case, the antigen is incubated with a specific antibody for this parameter and only recognizes and binds to this analyte. The product of this antigen-antibody reaction is a complex containing antibody-bound antigen. The higher the concentration of antigen in the patient sample, the higher the concentration of antigen-antibody complex formed. In current test systems, these antigen-antibody reactions are performed freely in solution (detected by turbidimetry or turbidimetry) or performed on an antigen specific surface (eg, RIA, ELISA). When the antigen-antibody reaction is performed, in the first case, the antigen-antibody complex is in solution, and in the second case, it is bound to the solid phase, mainly the plastic surface. The detection and quantification of the antigen-antibody complex is carried out by measuring the turbidity in the case of turbidity measurement or nephelometry, and in the case of RIA (radioimmunoassay), the radioisotope mark antibody is combined with the radiation detection. In the case of ELISA (enzyme immunoassay) and in the case of LIA, it is carried out with an enzyme-marked antibody together with the detection of an enzyme-catalyzed color reaction. By means of these test systems, analyte and antigen concentrations can be detected in the range of up to 1 pg / ml (protein).

The miniaturization and automation of the analysis system established in experimental technology is currently being intensively studied. For this reason, in recent years, various miniaturized solid-phase link test systems have been developed, and these systems are called “biochips” similar to computer chips because of their small size. The size of such a biochip is typically 0.25 to 9cm ^2. A biochip described in one document is a solid matrix of glass (S. P. A. Fadder et al .: Light-directed, spatially addressable parallel chemical synthesis; Science 251 (February 1991), 77-76. C. A. Outside Rowe: An array immunosensor for simulaneous detection of clinical analysts; Analytical Chemistry Col. 71 (January 15, 1999) p. 433-439; G. Mendoza et al: High-throughput microarray-based enzyme-linked immunosorbent assay (ELISA); BioTechniques 27 (October 1999), p. 778-788), nylon or nitrocellulose membrane, silicon resin (J. Cheng et al .: Chip PCR. Nucleic Acids Research Col. 24 (1996) p. 380-385), or silicon. It is also possible to covalently bind biomolecules such as DNA, peptides, proteins to these matrices through different linker molecules. A surface of 3.6 cm ² can be given up to 10,000 different biomolecules, which requires precise addressing of the biomolecules to specific areas of the matrix that are separated from each other. . This can be done, for example, by photolithographically controlled synthesis of biomolecules (S. P. A. Fadder et al .: Light-directed, spatially addressable parallel chemical synthesis; Science 251 (February 1991), 77-77. ) Or by using a precision mechanical, microprocessor-controlled programmable pipette robot (L. G. Mendoza et al .: High-throughput microarray-bassed enzyme-linked immunosorbent assay (ELISA)) BioTechniques 27 (October 1999), p. 778-788; M. Eggers et al .: A mi , and can be realized by: crochip for quantitative detection of molecular luminescent and radioisotope reporter groups; BioTechniques 17 (1994), p. 516-523).

  Using the solid phase bound biomolecule, the analyte to be analyzed contained in the patient sample can then be bound and detected with an appropriate detection system.

  As a highly sensitive detection system, for example, a charge-coupled device (CCD) camera (L. G. Mendoza, outside M. Eggers; the above-mentioned location), a phototransistor (T. Vo-Dinh outside: DNA biochip using a phototransistor integrated circuit). Analytical Chemistry 71 (1999) p. 358-363) and a fluorescence detector (outside SP A. Fodor; cited above) are used.

  The biochip is currently used only for research purposes, eg, DNA sequencing, gene discovery analysis, gene mutation analysis, and protein binding studies, ie antibody binding studies. Gene discovery analysis (M. Chen et al .: Quantitative monitoring of gene expression patterns with a complementary DNA microarray; DNA complementary to Science 270 (October 20, 1995), p. 467-470). Using a pipette robot, a spot blank is spotted on a chip blank matrix, and at this time, all of one spot represents a gene. Subsequently, the mRNA is separated from the tissue sample to be analyzed, marked with a fluorescent dye, and applied to the entire biochip. Next, the binding of the marked mRNA molecule to a site on the biochip can be detected. After the analysis is performed, the biochip is discarded because it is contaminated with the mRNA sample. Similarly, the biochip can be used for DNA sequence analysis and gene mutation analysis.

  Regarding the immunochemical analysis of sample materials, Mendoza et al. (Supra) described a prototype of a miniaturized test system. The author uses a special glass slide with 96 wells, and each well is printed with a pattern of spots containing 144 different antigens using a pipette robot and capillary printing method. After incubation with a solution containing the antibody, spots where antigen-antibody complexes are formed can be detected using a CCD camera.

  Thus, biochips known from the prior art are limited to embodiments in which specific, selected or specially synthesized molecules are applied and bound to a solid matrix in a defined and miniaturized arrangement. . These molecules are responsible for detecting the presence of an analyte in a mixture of heterogeneous components, eg, a patient sample. That is, these biochips only allow test designations, i.e., detection reactions predetermined by the detection molecules present on the chip.

  This approach has several disadvantages with respect to clinical diagnostic usage. With this approach, a number of different measurements can be performed simultaneously, but often has the problem that only a limited number of parameters, i.e., analytes are apparent. That is, the plurality of detection molecules present on a chip remain unused or uninteresting. Furthermore, it must be taken into account that such biochips should in principle not be used for further analysis after incubation with patient samples. For these reasons, the use of the above types of biochips is not well suited for routine detection methods in clinical testing and cannot be utilized in a cost effective manner.

  Furthermore, previously known biochips required new patient sample material for each further analysis. As a result, the patient has to travel several times to get a blood sample, which involves some effort on the doctor and each patient. If a later point-in-time test is to be repeated with a sample that has already been collected, the sample material will need to be stored intermediately in liquid or frozen state, with the above drawbacks.

  As a result of development, new detection reactions are always accessible. In order to be able to use biochips known from the prior art in such newly developed detection reactions, the biochip must always be updated, which involves high expenditures, It becomes possible with a delay in time.

  Sometimes, when new detection methods become available, one patient's original sample material is no longer available, and the long-term storage of the patient's sample material is a problem, examining changes in clinical parameters of interest over time There are many cases where this is not possible. Biochips known from the prior art cannot contribute to the solution of this problem.

  The work underlying the present invention is therefore to provide a miniaturized analysis system in the form of a biochip for diagnostic purposes, which is the same patient even for relatively long time intervals. It is possible to repeatedly perform the analytical detection method on a number of sample materials. In addition, biochips allow sample material to be stored and archived over a long period of time, saving space.

  Furthermore, the work that forms the basis of the present invention provides a method that allows the biochip to be subjected to a detection reaction automatically according to the requirements. This method must be very flexible, ie easily adaptable to other originally unintended detection reactions. In addition, the process allows repeated testing of the same sample material at time intervals.

  The problem can be solved by the biochip according to claim 1 and by the diagnostic detection process according to claim 17. The dependent claims relate to further embodiments of the invention that solve the problem as well.

  According to claim 1, the biochip according to the invention comprises a sample carrier consisting of a solid matrix to which sample material from a biological organism to be analyzed is linked to the surface. Thus, the sample material to be analyzed is linked to the solid phase.

  Compared to biochips known from the state of the art, in the case of the present invention no specifically selected or synthesized molecules are bound to the surface of the chip, but instead the sample material itself, eg different A body fluid containing a mixture of heterogeneous components of biomolecules is bound. Accordingly, the biochip according to the present invention is a patient-designated or patient sample-designated chip, and the known biochips so far are test-designated, ie, detection-designated biochips. The binding of the sample material may take place directly on the surface of the sample carrier matrix. The matrix or surface of the sample carrier is preferably pretreated by chemical or physical methods to improve the binding capacity of the surface to the sample material. Suitable methods for this purpose are known to the person skilled in the art, for example etching or roughing.

  The general structure of a biochip according to the present invention can be seen from FIG. 1A, which is a cross-sectional view of a biochip (a) comprising a sample carrier (b) and sample material (2) coupled thereto. The sample carrier consists essentially of a solid matrix (1).

  The binding of the sample material may optionally be performed by means of a layer of linker molecules (linker layer) applied to the surface of the sample carrier.

  The schematic structure of such a biochip (a) is shown in FIG. 1B, which shows a sample carrier (1) comprising a solid matrix (1) and a linker layer (3) located on the solid matrix. FIG. 2b is a cross-sectional view of sample material (2) bonded to the surface of the sample carrier via a linker layer.

  With the biochip of the present invention, the diagnostic detection reaction allows a molecule suitable for detection to be applied to a very small area of the biochip that is coated with the sample material, as described in detail below. Mainly done. In this way, it is possible to treat different areas of the surface of the biochip with different diagnostic reagents simultaneously or sequentially.

  In the biochip of the present invention, the sample material to be analyzed, rather than the molecules used for detection, is bound to the surface of the sample carrier that forms the matrix of the biochip, and the use and field of application of such chips is Is not limited to certain selected diagnostic molecules. Rather, the selection of useful or necessary diagnostic detection reagents is made only when the detection method is applied, thus making the application very flexible.

  In the biochip according to the invention, the sample material to be analyzed is bound to the surface of the sample carrier. Since the sample material is in a dry state, it is stable during storage even at room temperature. Because the biochip of the present invention is a miniaturized system, patient sample material can be saved and archived in a space-saving manner. In this way, a large number of patient-specific biochips according to the present invention can be stored in a small area and can be used to perform a number of analytical detection reactions.

  The possibility of archiving biochips, along with the possibility of performing several detection reactions at various sites on the chip surface independently of each other, of specific diagnostic markers in a specific patient or a specific sample of patients Enable inspections at different time intervals. In particular, it is advantageous that such a diagnostic test can be performed at a later time, even if it was originally not planned at the time of sample collection. This applies in particular to detection reactions developed at a later time after collection.

  The individual samples of the biochip according to the present invention preferably comprise sample material collected from a respective individual biological organism, for example sample material from a patient. Further biological organisms that can obtain suitable sample material other than humans are animals, plants, fungi and microorganisms.

  Sample materials preferably used as the sample material linked to the surface of the biochip include, for example, whole blood, plasma, serum, urine, ascites, amniotic fluid, saliva, fluid, cleaning material from body cavity, or bronchoalveolar lavage. Body fluid. Similarly, a tissue sample or organ sample, or a component, cell, fraction, cytolytic material, concentrate or extract from the liquid or tissue or organ sample may be used. In some cases, it may be appropriate to pre-treat such that the analyte of interest is concentrated in the sample. For example, for serum analysis, the solid components of a blood sample are separated by centrifugation, and the serum thus obtained is subsequently applied and bound to the surface of the sample carrier.

In a particularly advantageous embodiment, the surface of the sample carrier according to the invention on which the sample material is placed is divided into micro regions. These micro-areas are very small surface areas that are placed close to each other but spaced from each other. Usually, the surface is subdivided into a plurality of such microregions, preferably at least 100 microregions per cm ² . For some applications, a relatively small number of micro-ranges per unit area, for example 10-100 micro-areas per cm ² is sufficient. The micro-zones may preferably be configured as depressions or may be separated from each other by a hydrophobic or insoluble boundary region. By providing hydrophobic or insoluble boundary regions on the sample carrier, it is possible to prevent linking of sample material in these regions, so that these boundary regions located between the individual boundary regions have no sample material in them. Absent. The structure of the micro area is further illustrated below as an example with reference to FIGS.

  By dividing the chip surface into micro regions, the detection and treatment of a single site by the pipette robot is facilitated, thus improving the performance of the analytical detection reaction. In particular, if the micro-areas are provided in the form of a regular grid or screen, it is possible to identify and address individual micro-areas using their respective coordinates. This simplifies the selective application of detection reagents to individual parts of the biochip using a pipette robot. At the same time, the section of the chip surface coated with the sample material improves the detection reliability and the risk of unwanted reaction or contamination, as individual micro-zones are separated from each other by boundary regions or in the form of depressions. Sex is prevented.

  The sample carrier of the biochip according to the invention, in which the sample material is bound to the surface, consists essentially of a solid matrix. Suitable base materials for the sample carrier matrix are, for example, silicon resins, plastics (eg polyvinyl chloride, polyester, polyurethane, polystyrene, polypropylene, polyethylene, ethylene polyterephthalate, polyamide, nitrocellulose) or glass. Other solid materials may be used. A transparent solid material is preferentially used. However, when selecting a matrix material, one must consider whether it is appropriate to ensure that the intended sample material is bonded. The sample carrier may be a stack of at least two different matrix materials, with one of the two layers preferably being a rigid carrier layer and the other being the surface that binds the sample material.

As already mentioned above, it is particularly advantageous if the surface of the sample carrier to which the sample material is linked is subdivided into micro-regions. The outer dimensions of the biochip according to the invention are chosen so that the requirements for miniaturization are met. The surface of such a biochip or of a sample carrier for forming such a biochip is at most 10 cm ² , preferably at most 4 cm ² , particularly preferably at most 1 cm ² .

  The sample carrier, and thus the biochip, is preferentially configured as a flat shape and particularly preferably has a square or rectangular profile. However, other geometric shapes such as round or circular sample carriers or biochips are also suitable. The thickness of the flat sample carrier is preferably less than 3 mm, particularly preferably less than 1 mm. In individual cases, the outer dimensions of the biochip are selected to be compatible with already available pipette robots or analytical automata and to allow the use of the biochip according to the invention in such instruments.

  To form the biochip of the present invention, the surface of the sample carrier is coated with a biological sample material, which is bound to the surface of the sample carrier, and this binding may be shared or non-shared. To improve the ability to bind sample materials, and therefore biomolecules, when certain matrix materials are used as sample carriers, chemical or physical methods (eg In some cases, it may be useful to improve the binding ability by etching. Furthermore, by chemical or physical pretreatment, the surface of the sample carrier can be enlarged, thus improving the binding capacity. Suitable methods for this purpose are known to those skilled in the art.

  According to a preferred embodiment of the present invention, to improve the binding of the sample material and thus the analyte (eg biomolecules or cells) contained therein to the surface of the sample carrier, or an analyte, Alternatively, a linker layer may be provided on the surface of the sample carrier prior to application of the sample material to produce a selective or preferred binding of analyte groups or classes. In this way, an enrichment or preselection of an analyte or group of analytes can be realized. In this connection, the linker can, on the one hand, bind tightly to the surface of the sample carrier, i.e. its matrix material, on the other hand, bind biological sample material, or bind biological sample material. It is understood to mean a compound that can be bound selectively or in a preferred manner.

  Preferably, the surface of the sample carrier is preferably the selective binding or enrichment of a group of biological macromolecules such as proteins, peptides, glycoproteins, sugars, lipids, nucleic acids, or cells or A layer of linker molecules is provided that allows binding or enrichment of types or cell populations. Furthermore, it may be advantageous to coat different areas of the surface of the sample carrier with different types of linker molecules. In this way, for example, pre-selection and spatial separation of analytes (eg, different classes of biological macromolecules) present in a patient sample can be performed.

  Linker molecules include, for example, (L. C. Sliver-Lake; Antibacterial Immobilization using heterocrosslinkers; Binding amino acids), 4- (N-maleimidomethyl) -cyclohexane-1-carboxylhydrazide-HCl (M2C2H; for binding residual sugars), or binding the most different macromolecules or cells or cell types There are antibodies with known binding properties.

As already mentioned, it is advantageous if the surface of the sample carrier or biochip is divided into micro regions. Other than the methods already described, this partitioning may be realized by a linker layer constructed intermittently. In this case, it is preferable that the micro area of the surface area including the linker and the zone without the linker between them are formed, and there is at least 100 micro area per 1 cm ² .

  The manufacture of a sample carrier having a surface that is optimal for binding sample material and preferably subdivided into the micro-zone can be achieved in various ways. In order to form surface pieces of dimensions suitable for biochips, normally large matrix sheet raw materials (glass, plastic, etc.) can be handled in a corresponding manner using known mechanical methods. Separated, thereby obtaining a sample carrier. Alternatively, the corresponding matrix raw material is liquefied and subsequently cast into a suitable mold, for example by injection molding, to form a sample carrier.

  The described micro-regions, and therefore the intermediate boundary regions, can be obtained using physical methods such as engraving, punching, embossing or printing, or by chemical methods such as adding etching or hydrophobic layers. Can be formed. Furthermore, it is also possible to provide a linker layer in the form of a micro region on the surface of the sample carrier using a pipette robot. In addition, various known printing methods can be adapted to enable the formation of the microzones of the present invention. Finally, the methods described above may be used in combination. Furthermore, it is possible to subdivide into micro-areas which are already sheet-like matrix raw materials, which are then separated into individual sample carriers as described above.

  The biochip according to the present invention is particularly suitable for archiving sample material from one specific patient (“patient-designated” biochip) per biochip and performing multiple diagnostic analyses. In order to facilitate or enable such automatic processing and evaluation in chip archiving and analysis automata, according to a further embodiment of the invention, the biochip or corresponding sample carrier can be Alternatively, it is proposed that a means is provided that allows storage of patient-related data or analysis data. This can preferably be done using a machine readable bar code, a machine readable magnetic strip, a digital storage element, or another computer readable storage medium. The above embodiment is advantageous when a large number of patient-related biochips must be archived or processed.

  By having a means of storing data on the biochip, for example, a physician or clinician who collects the sample and potentially applies sample material to the sample carrier can, for example, store patient-related data or samples on the biochip. Data related to collection can be saved. Furthermore, it is possible to save the analysis results or their evaluations, whereby information about the analyzes already performed before can also be saved.

  The present invention further comprises a diagnostic detection method comprising a processing step of producing a biochip using a sample material and a step of performing and evaluating a diagnostic detection reaction.

  Initially, bodily fluids, tissue samples, etc. are obtained from the patient or another organism and are subjected to pretreatment (eg, centrifugation, concentration, etc.) if necessary. If necessary, the sample material is suspended or dissolved in a suitable buffer or solvent. In order to make a biochip, this sample material, preferably in liquid form, is applied to the surface of the sample carrier according to the invention, for example by pipetting or by dipping. After a short incubation time of several minutes, eg 1 to 10 minutes, all excess sample material is removed (eg by aspiration). Thereafter, it is preferably dried at a slightly elevated temperature (30-60 ° C.), whereby the sample material and thus the analyte (eg biomolecule) contained therein is linked to the surface of the sample carrier, Or, in some cases, it is linked to a linker layer. The foregoing processing steps are performed by a physician collecting sample material or by auxiliary medical staff. Thus, the resulting patient-designated or sample-designated biochip can then be stored dry and stored at room temperature, ready for analysis immediately or later.

  In order to carry out an analytical detection reaction on such a biochip coated with a sample material, the desired detection reagent is preferably placed in an individual site or individual micro area of the biochip at a later stage. Is given using. Suitable as detection reagents are, for example, antibodies, antigens, sequence specific antibodies, lectins, DNA probes, small molecule ligands, hormones, biomolecule binding dyes, or other specific binding molecules.

  The described site or micro-range of the biochip is incubated at a temperature for a period of time, where the selected detection reagent is at a concentration and the experimental parameters in each case are the type of detection reagent used and the sample Depends on the material. Those skilled in the art will usually be familiar with the selection of appropriate experimental parameters.

  After the determined incubation time, the liquid detection reagent is aspirated and removed using a pipette robot. Thereafter, in the next stage, using a pipette robot in the same manner, a washing solvent, and if necessary, further detection reagents are applied in sequence to the site or micro area to be analyzed and aspirated. Furthermore, the washing solutions and detection reagents used, such as fluorescently marked antibodies and suitable reaction conditions are in principle known to the person skilled in the art.

  Finally, the site or micro area where a positive detection reaction was seen is identified by an appropriate detector. Registration of these measurement signals is preferably realized using a CCD camera, a phototransistor, or a radioactivity, luminescence or fluorescence detector, and the selection of the detection method depends on the type of detection reagent used. Dependent. The main measurement data supplied by the detector can be subjected to computer aided evaluation. A positive detection reaction in principle forms an antigen-antibody complex, for example, by a specific interaction or linkage between the analyte (eg an antigen in a patient sample) and the detection reagent used. It happens. These can be detected by further reaction steps or reagents known to those skilled in the art.

  For example, if a relatively large number of biochips according to the invention from different patients are analyzed simultaneously or in parallel in the manner described, that is, detection reactions and detections are carried out on a plurality of biochips simultaneously or in parallel. This is particularly advantageous.

  It is further advantageous when a biochip or a plurality of biochips are treated in parallel (simultaneously) or subsequently sequentially, where different detection reagents have different detection specificities, in each case the detection reagent Applies to uninspected sites or micro areas. In this way, it is possible to test the presence or absence of diagnostic marker molecules with a plurality of patient-designated biochips in a very short time. In order to prevent a certain part or micro area of the biochip from being used for the detection reaction many times, the position of the "used" part or area is registered and stored in the biochip storage medium. Also good.

  Furthermore, it is possible to analyze different sites or micro-ranges of the same biochip, possibly using different concentrations of the same detection reagent. In this way, the reliability of detection is improved by multiple or parallel measurements.

  After the detection reaction and detection has taken place, the biochip can be returned for archiving and storage, making it available for further detection reactions. That is, the same biochip coated with a sample material of a patient can be subjected to analysis including detection reaction, detection and evaluation continuously two to several times according to the above steps, and the biochip is continuously Stored or archived during the time between analysis. In continuous analysis, different microregions of the biochip are treated with different detection reagents.

  Due to the possibility of performing multiple diagnostic detection reactions on a single biochip, even at relatively long time intervals, the amount of sample material required is significantly reduced, i.e. a relatively small amount (e.g. , 0.5 ml) based on the first sample, a biochip can be formed, allowing multiple detection reactions over a long period of time.

  The biochip according to the invention can be stored at room temperature for several years, at least 5 years, with the raw material bound to it, and can be used repeatedly to perform the detection reactions described above during this storage period. The biochip of the invention is therefore advantageous in all cases where it is important to store or archive sample material from a patient that gives the possibility to perform a diagnostic detection reaction.

  In special cases where the stability of an analyte in the sample material may be important, the biochip is stored at a low temperature, eg, 0-15 ° C, or at a relatively low temperature, eg, 0 ° C or less. May be. In principle, it is expected that large inactivation of potentially degraded enzymes such as nucleolytic or proteolytic enzymes will occur after long-term storage of the biochip in the dry state or after storage for several years. The

  The biochip of the present invention is conveniently stored or archived in an openable case or box with a guide rail along the inner side wall, along which the biochip is placed in the case. Locked. By providing a plurality of such guide rails, 100 biochips can be accommodated in a single case in a space-saving manner. Many such cases can be integrated as stationary or movable components in a drawer system or cupboard system.

  The biochip according to the present invention, and thus the diagnostic detection method according to the present invention, is suitable for a plurality of practical applications due to its storage stability and the possibility of carrying out a large number of individual detection reactions. In the following, some possible applications are described.

  The biochip according to the invention can be used for monitoring in the field of tumor diagnosis, for example for the appearance of certain tumor markers in serum or tissue samples. These tumor markers are formed by the tumor and secreted in the body fluid of the sick or formed by the organism as a result of a response to the tumor. Conversely, these marker molecules are not present in the serum or other body fluids of healthy people, or are present in trace amounts. As the tumor grows, the serum concentration of the marker increases. However, the assessment of “intermediate” serum concentrations refers to both abnormal normal values (eg, genetic causes) and the onset of tumor growth, despite being harmless to look for, and thus establish a diagnosis Problems arise in doing so. Thus, the definitive question is whether there has been an increase in the serum concentration of tumor markers over a period of time. This question is answered when the serum concentration of the tumor marker is tested at a certain time interval. Only in this way it is possible to recognize the onset of tumor growth relatively accurately at an early point. In principle, such marker detection must be performed for each test person at time intervals (ie, sequential determination) so that trend analysis can be performed. Trend analysis or sequential determination of tumor markers using the biochip of the present invention may be utilized for tumor development control or follow-up, for example, for post-surgical recurrence control or cytostatic therapy control.

  Using known archiving methods for patient samples, the test series described above cannot be implemented, or can be implemented only at great expense. In contrast, with the biochip according to the invention and the method according to the invention, this sample material can be stored in large quantities in a place-saving manner over a long period of time and a plurality of diagnostics, for example to determine tumor markers. It becomes possible to implement the detection method. This enables retrospective trend analysis that considerably facilitates the establishment of diagnosis. The archivability of the biochip according to the present invention is also possible when a new tumor marker has been known to test a proband's relatively old biochip for the presence or absence of a tumor marker. The present invention therefore contributes to recognizing tumor growth at an early stage and also contributes to appropriate initial intervention.

  Furthermore, the biochip of the present invention is suitable for clinical studies where archiving patient sample material is equally important. This is particularly noticeable, for example, if the scientific problem formulation is changed and it is necessary to check the previous sample of the patient later. Compared to the currently used method, ie a method in which a sample (in principle at least 0.5 ml) is frozen and later thawed, the biochip according to the invention enables and requires a considerable number of detection reactions. It is much more appropriate to allow for easy storage with much less space. Since more detection reactions are possible with one biochip, there is no longer the need to prepare and store several parallel samples of a patient at a particular time.

  A further field of application of the biochip according to the invention is to have human donor material (eg body fluids, cells, tissue), a sample of the donor material and possibly the product from it for diagnostic detection for control purposes A blood bank or farm or other mechanism that must be stored or must be performed to carry out the reaction. Usually, it takes up too much space, so only the analysis results are stored, not the sample itself. However, in connection with the occurrence of contamination, it is also advantageous to analyze such samples again, possibly using other detection methods, for example for legal safeguarding measures. Such a new analysis of the sample is also advantageous if the raw materials, intermediate materials, or end products of the human material are proven to comply with the legal provisions or conditions with respect to their composition. The biochip according to the present invention may be advantageously used for subsequent analysis of donor blood or blood banks, companies, and other donor materials from other mechanisms. If contaminated or suspected of being incompatible, the archived biochip will allow later analysis of suspected donor samples or product samples based on it. In this way, it is possible for the donor sample or product sample to later submit evidence that complies with the rules, standards or conditions of the law. This can be particularly important and helpful in legal disputes.

  Furthermore, the biochip or sample carrier of the present invention may be used for the storage and archiving of patient sample material for purposes of routine diagnosis of patient samples.

  The biochip according to the invention can be advantageously used in epidemiological studies, for example to examine the growth of infectious diseases. So far, such tests have often failed because the sample material from before was no longer available for practical reasons and therefore could not store the required number of blood samples. In contrast, the described biochip saves space and allows for long-term storage and archiving economically. Thereby, a large number of samples can be archived. These samples are utilized for later epidemiological studies, and therefore these studies can return to the archived samples from before, allowing for a larger set of patients.

  The biochip and method according to the present invention can be advantageously used in connection with different formulations of interest in the field of pharmacy or zoology. In addition to the application examples already described, application for the purpose of histocompatibility testing or application in the field of forensic medicine is also possible.

Figure 2 is a cross-sectional view through a biochip (a) according to the present invention comprising a sample carrier (b) and sample material (2) bonded to its surface. Fig. 2 is a cross-sectional view of an embodiment of a biochip (a) according to the present invention in which a sample carrier (b) has a solid matrix (1) and further a linker layer (3) located thereon. (B) is a cross-sectional view through the biochip in the region of the micro region (c), assuming that the sample carrier comprises the solid matrix (1). It is sectional drawing which passes along the biochip in the area | region of a micro area | region (c) similarly to FIG. 2A. It is sectional drawing which passes along the biochip in the area | region of a micro area | region (c) similarly to FIG. 2A. An embodiment as shown in FIG. 2A is shown. Fig. 2B shows a further embodiment of a display as in Fig. 2A. 2E shows a modification of the embodiment shown in FIG. 2E. 2E shows a modification of the embodiment shown in FIG. 2E. A modification of the embodiment shown in FIG. 2F is shown. 2 shows the basic shape of a biochip or sample carrier (b) according to the invention. A modification of the sample carrier (b) shown in FIG. 3A is shown. It is a top view which shows a sample carrier (b) or a biochip. A modification of the embodiment shown in FIG. 3C is shown.

  In the following, the basic principle and by way of example, several preferred embodiments of the invention will be described in more detail with reference to FIGS. However, the invention is not limited to the illustrated embodiments. Reference symbols are used throughout the drawings to refer to similar components, and each symbol shall have the same meaning.

  FIG. 1A is a cross-sectional view through a biochip (a) according to the present invention comprising a sample carrier (b) and sample material (2) bonded to its surface. The sample carrier (b) consists essentially of a solid matrix (1).

  FIG. 1B is a cross-sectional view of an embodiment of a biochip (a) according to the present invention in which the sample carrier (b) has a solid matrix (1) and further a linker layer (3) located thereon. The sample material (2) is linked to the matrix via the linker layer (3) of the sample carrier.

  FIG. 2 is a cross-sectional view of a different embodiment of the biochip according to the present invention including the above-mentioned micro area (c). In each of FIGS. 2A to 2H, a biochip region having a micro region is shown.

  FIG. 2A is a cross-sectional view through the biochip in the region of the micro region (c), where (b) represents a sample carrier comprising a solid matrix (1). As can be seen, the biological sample material (2) is bound to the surface of the sample carrier only in the region of the micro region (c). There is no sample material in the adjacent region of the sample carrier surface.

  FIG. 2B is a cross-sectional view through the biochip in the micro-region (c) as in FIG. 2A, but the sample material (2) passes through the linker layer (3) through the matrix (1) of the sample carrier (b). ). The linker layer exists only in the region of each micro region, and there is no linker molecule in the adjacent region.

  FIG. 2C is a cross-sectional view through the biochip in the micro area (c) as in FIG. 2A. Here, however, there are provided hydrophobic regions or non-soluble boundary regions (4) surrounding each micro-zone, in which no sample material (2) can be bound.

  FIG. 2D shows an embodiment as shown in FIG. 2A, in which the measures shown in FIGS. 2B and 2C are combined. The sample material (2) is linked to the matrix of the sample carrier via a linker layer (3) located in the region of the micro region (c). In addition, a hydrophobic characteristic or non-soluble boundary region (4) surrounding each micro region is provided.

  FIG. 2E shows a further embodiment of a display as in FIG. 2A. Here, a micro area (c) is shown, which is constituted in the form of depressions (5) in which the sample material (2) is bound to the matrix (1) of the sample carrier (b).

  FIG. 2F shows a variation of the embodiment shown in FIG. 2E, where the bottom is coated with a linker layer (3) that binds the sample material (2) to the matrix (1) of the sample carrier (b). The micro area (c) configured in the form (5) is shown.

  FIG. 2G shows a variation of the embodiment shown in FIG. 2E, where the recess (5) in the micro area (c) is configured in the form of a rounded groove.

  FIG. 2H shows a variation of the embodiment shown in FIG. 2F, where the recess (5) in the micro area (c) is configured in the form of a rounded groove.

  FIG. 3 is a plan view showing by way of example several embodiments of a biochip or sample carrier according to the present invention.

  FIG. 3A shows the basic shape of a biochip or sample carrier (b) according to the invention, where the surface area to be coated with the sample material is indicated by (6). The area outside the surface area (6), in particular the border area, may be hydrophobic or insoluble.

  FIG. 3B shows a modification of the sample carrier (b) shown in FIG. 3A, which further includes a bar code, a magnetic strip, or other storage medium (8).

  FIG. 3C is a plan view showing the sample carrier (b) or the biochip, and the arrangement of the micro regions (c) is shown as an example. As shown in FIGS. 3A and 3B, the boundary region is also hydrophobic or insoluble in this figure.

  Finally, FIG. 3D shows a variation of the embodiment shown in FIG. 3C, and as shown in FIG. 3B, further means for storing data are provided, such as a bar code, a magnetic strip, or another storage medium. .

Claims (25)

A biochip for diagnostic purposes having a solid matrix sample carrier, the surface of the sample carrier being subdivided into micro-regions, to which sample material originating from individual organisms to be analyzed is bound The sample material is a body fluid, tissue sample or organ sample, or the sample material is a component, cell, fraction, concentrate from the body fluid, tissue sample or organ sample described above. Alternatively , a biochip consisting of an extract, wherein the individual organism is a human, animal, plant, or fungal organism, or a microorganism. The biological sample material is directly bonded to the surface of the matrix of the sample carrier, and the matrix or surface of the sample carrier is chemically or chemically modified to improve the binding capacity of the surface to the sample material. The biochip according to claim 1, wherein the biochip is pretreated by a physical method. The biochip according to claim 1 or 2, characterized in that a sample material from an individual patient is bound to the surface of each single sample of the biochip. The body fluid, whole blood, plasma, serum, urine, peritoneal fluid, amniotic fluid, saliva, liquid, cleaning material from the body cavity, or, characterized in that it is a bronchoalveolar lavage, either one of claims 1 to 3 The biochip according to one item. The surface of the sample carrier is characterized in that it is subdivided into at least 100 micro area per 1 cm ^2, the biochip of any one of claims 1 to 4. The biochip according to any one of claims 1 to 5 , characterized in that the micro-areas are configured as depressions or are separated from each other by a hydrophobic or insoluble boundary area. The surface area of the sample carrier is at most 10 cm ² , the sample carrier is configured as a flat body, and the thickness of the flat sample carrier is less than 3 mm. The biochip as described in any one of thru | or 6 . The sample surface area of the carrier is characterized in that it is at most 4 cm ^2, any one claim of the biochip of the claims 1 to 7. 9. The biochip according to any one of claims 1 to 8 , wherein the sample carrier is configured as a flat body having a square or rectangular outer shape. The biochip according to any one of claims 1 to 9 , wherein the sample carrier is configured as a flat body, and the thickness of the flat sample carrier is less than 1 mm. The matrix or the surface of the sample carrier has been pretreated in a chemical or physical manner, or the surface of the sample carrier has been enlarged by a chemical or physical pretreatment. A biochip according to any one of claims 1 to 10 , characterized in that it is characterized in that The biochip according to any one of claims 1 to 11 , wherein the surface of the sample carrier is coated with a linker layer, and the sample material is bonded using the linker layer. The linker layer allows selective binding or enrichment of specific groups of proteins, peptides, glycoproteins, sugars, lipids, nucleic acids, or selective binding of cells or certain cell types or cell populations The biochip according to claim 12 , characterized in that it is composed of a linker molecule that enables The linker layer on the surface of the sample carrier is characterized in that a zone free of micro-region and an intermediate linker intermittent become as Configured surface comprising a linker is formed, according to claim 12 or 13. The biochip according to 13 . The biochip according to any one of claims 1 to 14 , further comprising means for enabling identification of the biochip, storage of patient-related data, or storage of analysis data. The biochip of claim 1, wherein the sample material is prepared by centrifugation, cell disruption or extraction before being attached to the surface of the sample carrier. A method for using the biochip according to any one of claims 1 to 16 for diagnostic detection, comprising: a) a specific diagnostic detection reagent in one micro area of the biochip using a pipette robot And incubating under conditions suitable for the relevant detection reaction, b) applying a wash solution to inhibit unspecified reactions and subsequently removing the wash solution, and c) if necessary Repeating steps a) and b); d) detecting a measurement signal from a positively reacting micro-range; and e) computer-aided evaluation of the measurement data and storing the measurement data. Method. The method according to claim 17 , characterized in that the steps a) to e) are performed in parallel or simultaneously on one or more biochips. 18. A method according to claim 17 , characterized in that the individual microregions of the biochip are treated simultaneously or sequentially with different specific diagnostic detection reagents. Specific diagnostic detection reagents utilized in the above step a), an antibody, antigen, lectin, DNA probes, biomolecule-binding dyes or, characterized in that it is a other specific binding molecules, according to claim 17 Method. 18. The method according to claim 17 , wherein the measurement signal is detected using a CCD camera, a phototransistor, or a radioactivity, fluorescence or luminescence detector. The same biochip coated with a patient's sample material is analyzed according to the above steps a) to d) or the above steps a) to e) in succession twice or several times. 18. A method according to claim 17 , characterized in that it is preserved during the period between successive analyses, and that different microregions of the biochip are treated with detection reagents in the successive analyses. Diagnostic detection for storing and archiving and / or the sample material collected from a patient for the reaction, use of a biochip as claimed in any one of claims 1 to 16. For purposes of routine diagnosis of patients' samples, clinical studies, or epidemiological studies, for blood banks, companies, or other mechanisms, or for purposes of quality assurance and quality confirmation in tumor diagnosis, or storing and archiving patient sample material for trial purposes, the use of biochips as claimed in any one of claims 1 to 16. For pharmaceutical or zoological purposes, for tumor diagnosis, for tumor marker analysis in clinical studies, for quality assurance in blood banks, companies or other institutions, for epidemiological studies, for tissue typing, or Use of a biochip according to any one of claims 1 to 16 for trial purposes.

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